1. Field of the Invention
This invention relates in general to the controlled dispensing of small volumes of liquid, and more particularly, to precisely metering the volume of liquid dispensed by a fluid microdispenser. Even more particularly, this invention relates to reproducibly controlling sub-nanoliter liquid drop size in a fluid microdispenser.
2. Description of the Related Art
Dispensing liquid volumes of less than 1 nanoliter accurately and reproducibly in a single drop is a long-sought goal in areas as diverse as chemical screening for drug discovery, pharmaceutical formulation, agricultural chemistry, cosmetic and food processing, and ink-jet printing. In drug discovery, for example, small quantities of chemical substances dissolved in liquid at large concentration are distributed to a large number of reaction wells each with a volume capacity of 1 μl, in which a biological assay is replicated many times. These concentrates include test chemical compounds with unknown biochemical or physiological effects in which it is desired to construct many reactions with the same concentration of the test chemical compound in each reaction.
Precise metering is also useful in analytical chemistry to distribute small quantities of concentrates of fluorimetric or radiometric indicator compounds used to measure the rate and extent of a chemical, biochemical, or physiological reaction. In cell culture, it is desired to deliver small quantities of valuable biological reagents necessary for the survival of cells or tissue explants cultured to provide a platform for biological assays. In large-format automated arrays of liquid dispensers in which the intrinsic drop volume is different from dispenser to dispenser, it is desirable to adjust the drop volume delivered by each dispenser so that all dispensers are “tuned” to deliver droplets of identical or nearly identical volume.
In all these applications, the crucial demand is delivery of sub-nanoliter liquid drops in which the volume of each drop is identical (or nearly so) and can be adjusted to the needs of the application. Many designs for dispensers have been utilized for producing sub-nanoliter-volume drops. In many circumstances, a piezoelectric actuator is coupled to a liquid filled tube that contains a circular orifice at one end from which liquid drops are ejected. When the piezoelectric material is actuated by an electrical voltage pulse, the piezoelectric material increases in thickness and compresses the liquid-filled tube by decreasing its volume. This compression induces a pressure increase in the liquid that travels throughout the interior of the tube to the liquid-vapor interface that spans the orifice at the dispensing end of the tube. If the magnitude of the pressure is sufficient to overcome the forces that limit the formation of a liquid drop, such as the interfacial tension required to increase the area of liquid surface in contact with air, the viscous drag of pressure-driven liquid movement, and the inertia inherent in causing a mass of liquid to move, then a liquid drop is ejected from the orifice. Such systems are described in U.S. Pat. No. 3,683,212 to Zoltan, U.S. Pat. No. 3,946,398 to Kyser et al, and U.S. Pat. No. 4,877,745 to Hayes et al, which are hereby incorporated herein by reference in their entirety.
These methods of liquid dispensation involve several complications. Firstly, there are several modes of drop formation. When the piezoelectric element is actuated with a voltage pulse of low amplitude, drop formation is intermittent, in that not every actuation pulse elicits ejection of a single drop. Identically sized pulses may elicit drops with different volumes. With an actuation pulse of large amplitude, a large volume of liquid may be ejected from the orifice, resulting in the formation of multiple drops for each pulse (such as satellites). These drops may have different trajectories, resulting in the possibility that some of the ejected liquid may miss its desired target. In between these small and large actuation pulse amplitudes is a range over which each pulse elicits dispensing of a single drop that is identical upon each actuation. As the pulse amplitude is increased or decreased, the volume of the ejected drop is increased or decreased in proportion. This uniform mode of dispensing is most desired when it is imperative to deliver a fixed quantity of liquid to the same location on each actuation.
A further complication with these systems is the shape of the lumen of the fluid reservoir. The choice of fluid reservoir lumen diameter is determined by many factors. These factors may include the need for a low hydraulic resistance to facilitate the movement of system fluid and sample liquid into and out of the fluid reservoir for washing as well as the expense and ability to create a lumen of desired uniform diameter and smoothness. In addition, a larger lumen will prevent obstruction by the aggregation of solid or colloidal material that may be present in the sample. The diameter of the orifice, however, is selected on the basis of the desired drop volume, which usually scales as volume−(diameter)3 (Hayes et al). Therefore, to eject drops with volumes on the order of less than 1 nl requires an orifice diameter less than 100 μm. Since lumenal diameters of the fluid reservoir may approach 1 mm or greater, there is often a mismatch in diameter of the components in the pathway along which the actuation pressure is transmitted.
The way that this mismatch is accommodated in a dispenser may determine the effectiveness of the actuation pulse. For example, in the piezo dispensers of Bogy and Talke and Zoltan, the orifice was drilled through a plate that was then cemented over one end of a 1 mm-diameter tube reservoir. These dispensers required voltage pulses across the piezoelectric elements in excess of 300 V to actuate drop ejection. Other methods to create a taper in the lumen of the tube include heating a small region of a glass tube and then drawing the tube so that the lumen narrows to the necessary orifice diameter. However, this type of heating-pulling method may result in a variable change in radius as a function of longitudinal distance down the tube as the orifice is approached, so that each drawn tube may have a different taper shape and hence, different dispensing characteristics.
The taper shape in turn influences the hydrodynamic mechanism of the pump. To form a nozzle, the tube lumen narrows and terminates as an orifice of diameter less than the diameter of the tube lumen in its straight portion. Where the tube radius begins to decrease, the fluid stream turns toward the nozzle. Restriction to flow in the longitudinal direction creates flow in the radial direction due to the buildup of a pressure gradient in the radial direction. This radial gradient of pressure has the effect of decreasing the longitudinally directed pressure gradient. If the longitudinal pressure gradient is decreased too much, then it will be insufficient to push enough liquid out the orifice to create a drop.